Plasmodium falciparum AMA-1 protein and uses thereof

ABSTRACT

In this application is described the expression and purification of a recombinant  Plasmodium falciparum  (3D7) AMA-1 ectodomain. The method of the present invention produces a highly purified protein which retains folding and disulfide bridging of the native molecule. The recombinant AMA-1 is useful as a diagnostic reagent, for use in antibody production, and as a vaccine.

[0001] The present invention also relates to a method for in vitrodiagnosis of malaria antibodies present in a biological sample,comprising at least the following steps

[0002] (i) contacting said biological sample with a compositioncomprising any of the AMA1/E peptides as defined above, preferably in animmobilized form under appropriate conditions which allow the formationof an immune complex, wherein said peptide or protein can be abiotinylated peptide or protein which is covalently bound to a solidsubstrate by means of streptavidin or avidin complexes,

[0003] (ii) removing unbound components,

[0004] (iii) incubating the immune complexes formed with heterologousantibodies, with said heterologous antibodies having conjugated to adetectable label under appropriate conditions,

[0005] (iv) detecting the presence of said immune complexes visually ormechanically (e.g. by means of densitometry, fluorimetry, colorimetry).

[0006] The present invention also relates to a kit for determining thepresence of malaria antibodies, in a biological sample, comprising:

[0007] at least one peptide or protein composition as defined above,possibly in combination with other polypeptides or peptides fromPlasmodium or other types of malaria parasite, with said peptides orproteins being preferentially immobilized on a solid support, morepreferably on different microwells of the same ELISA plate, and evenmore preferentially on one and the same membrane strip,

[0008] a buffer or components necessary for producing the bufferenabling binding reaction between these polypeptides or peptides and theantibodies against malaria present in the biological sample,

[0009] means for detecting the immune complexes formed in the precedingbinding reaction,

[0010] possibly also including an automated scanning and interpretationdevice for inferring the malaria parasite present in the sample from theobserved binding pattern.

[0011] The immunoassay methods according to the present inventionutilize AMA1/E domains that maintain linear (in case of peptides) andconformational epitopes (proteins) recognized by antibodies in the serafrom individuals infected with a malaria parasite. The AMA1/E antigensof the present invention may be employed in virtually any assay formatthat employs a known antigen to detect antibodies. A common feature ofall of these assays is that the antigen is contacted with the bodycomponent suspected of containing malaria antibodies under conditionsthat permit the antigen to bind to any such antibody present in thecomponent. Such conditions will typically be physiologic temperature, pHand ionic strenght using an excess of antigen. The incubation of theantigen with the specimen is followed by detection of immune complexescomprised of the antigen.

[0012] Design of the immunoassays is subject to a great deal ofvariation, and many formats are known in the art. Protocols may, forexample, use solid supports, or immunoprecipitation. Most assays involvethe use of labeled antibody or polypeptide; the labels may be, forexample, enzymatic, fluorescent, chemiluminescent, radioactive, or dyemolecules. Assays which amplify the signals from the immune complex arealso known; examples of which are assays which utilize biotin and avidinor streptavidin, and enzyme-labeled and mediated immunoassays, such asELISA assays.

[0013] The immunoassay may be, without limitation, in a heterogeneous orin a homogeneous format, and of a standard or competitive type. In aheterogeneous format, the polypeptide is typically bound to a solidmatrix or support to facilitate separation of the sample from thepolypeptide after incubation. Examples of solid supports that can beused are nitrocellulose (e.g., in membrane or microtiter well form),polyvinyl chloride (e.g., in sheets or microtiter wells), polystyrenelatex (e.g., in beads or microtiter plates, polyvinylidine fluoride(known as Immunolon.TM.), diazotized paper, nylon membranes, activatedbeads, and Protein A beads. For example, Dynatech Immunolon.TM.1 orImmunlon.TM. 2 microtiter plates or 0.25 inch polystyrene beads(Precision Plastic Ball) can be used in the heterogeneous format. Thesolid support containing the antigenic polypeptides is typically washedafter separating it from the test sample, and prior to detection ofbound antibodies. Both standard and competitive formats are know in theart.

[0014] In a homogeneous format, the test sample is incubated with thecombination of antigens in solution. For example, it may be underconditions that will precipitate any antigen-antibody complexes whichare formed. Both standard and competitive formats for these assays areknown in the art.

[0015] In a standard format, the amount of malaria antibodies in theantibody-antigen complexes is directly monitored. This may beaccomplished by determining whether labeled anti-xenogeneic (e.g.anti-human) antibodies which recognize an epitope on anti-malariaantibodies will bind due to complex formation. In a competitive format,the amount of malaria antibodies in the sample is deduced by monitoringthe competitive effect on the binding of a known amount of labeledantibody (or other competing ligand) in the complex.

[0016] Complexes formed comprising anti-malaria antibody (or in the caseof competitive assays, the amount of competing antibody) are detected byany of a number of known techniques, depending on the format. Forexample, unlabeled malaria antibodies in the complex may be detectedusing a conjugate of anti-xenogeneic Ig complexed with a label (e.g. anenzyme label).

[0017] In an immunoprecipitation or agglutination assay format thereaction between the malaria antigens and the antibody forms a networkthat precipitates from the solution or suspension and forms a visiblelayer or film of precipitate. If no anti-malaria antibody is present inthe test specimen, no visible precipitate is formed.

[0018] There currently exist three specific types of particleagglutination (PA) assays. These assays are used for the detection ofantibodies to various antigens when coated to a support. One type ofthis assay is the hemagglutination assay using red blood cells (RBCs)that are sensitized by passively adsorbing antigen (or antibody) to theRBC. The addition of specific antigen antibodies present in the bodycomponent, if any, causes the RBCs coated with the purified antigen toagglutinate.

[0019] To eliminate potential non-specific reactions in thehemagglutination assay, two artificial carriers may be used instead ofRBC in the PA. The most common of these are latex particles. However,gelatin particles may also be used. The assays utilizing either of thesecarriers are based on passive agglutination of the particles coated withpurified antigens.

[0020] The AMA1/E proteins, peptides, or antigens of the presentinvention will typically be packaged in the form of a kit for use inthese immunoassays. The kit will normally contain in separate containersthe AMA1/E antigen, control antibody formulations (positive and/ornegative), labeled antibody when the assay format requires the same andsignal generating reagents (e.g. enzyme substrate) if the label does notgenerate a signal directly. The AMA1/E antigen may be already bound to asolid matrix or separate with reagents for binding it to the matrix.Instructions (e.g. written, tape, CD-ROM, etc.) for carrying out theassay usually will be included in the kit.

[0021] Immunoassays that utilize the AMA1/E antigen are useful inscreening blood for the preparation of a supply from which potentiallyinfective malaria parasite is lacking. The method for the preparation ofthe blood supply comprises: reacting a body component, preferably bloodor a blood component, from the individual donating blood with AMA1/Eproteins of the present invention to allow an immunological reactionbetween malaria antibodies, if any, and the AMA1/E antigen, anddetecting whether anti-malaria antibody-AMA1/E antigen complexes areformed as a result of the reacting. Blood contributed to the bloodsupply is from donors that do not exhibit antibodies to the AMA1/Eantigens.

[0022] The present invention further contemplates the use of AMA1/Eproteins, or parts thereof as defined above, for in vitro monitoringmalaria infection or prognosing the response to treatment (for instancewith chloroquine, mefloquine, Malarome) of patients suffering frommalaria infection comprising:

[0023] incubating a biological sample from a patient with malariainfection with an AMA1/E protein or a suitable part thereof underconditions allowing the formation of an immunological complex,

[0024] removing unbound components,

[0025] calculating the anti-AMA1/E titers present in said sample (forexample at the start of and/or during the course of therapy),

[0026] monitoring the natural course of malaria infection, or prognosingthe response to treatment of said patient on the basis of the amountanti-AMA1/E titers found in said sample at the start of treatment and/orduring the course of treatment.

[0027] Patients who show a decrease of 2, 3, 4, 5, 7, 10, 15, orpreferably more than 20 times of the initial anti-AMA1/E titers could beconcluded to be long-term, sustained responders to malaria therapy.

[0028] It is to be understood that smaller fragments of theabove-mentioned peptides also fall within the scope of the presentinvention. Said smaller fragments can be easily prepared by chemicalsynthesis and can be tested for their ability to be used in an assay asdetailed above.

[0029] The present invention also relates to a kit for monitoringmalaria infection or prognosing the response to treatment (for instanceto medication) of patients suffering from malaria infection comprising:

[0030] at least one AMA1/E peptide as defined above,

[0031] a buffer or components necessary for producing the bufferenabling the binding reaction between these proteins or peptides and theanti-AMA1/E antibodies present in a biological sample,

[0032] means for detecting the immune complexes formed in the precedingbinding reaction,

[0033] possibly also an automated scanning and interpretation device forinferring a decrease of anti-AMA1/E titers during the progression oftreatment.

[0034] The present invention also relates to a serotyping assay fordetecting one or more serological types or alleles of malaria parasitepresent in a biological sample, more particularly for detectingantibodies of the different types or alleles of malaria parasites to bedetected combined in one assay format, comprising at least the followingsteps:

[0035] (i) contacting the biological sample to be analyzed for thepresence of malaria antibodies of one or more serological types, with atleast one of the AMA1/E compositions as defined above, preferentially inan immobilized form under appropriate conditions which allow theformation of an immune complex,

[0036] (ii) removing unbound components,

[0037] (iii) incubating the immune complexes formed with heterolagousantibodies, with said heterologous antibodies being conjugated to adetectable label under appropriate conditions,

[0038] (iv) detecting the presence of said immune complexes visually ormechanically (e.g. by means of densitometry, fluorimetry, calorimetry)and inferring the presence of one or more malaria serological typespresent from the observed binding pattern.

[0039] It is to be understood that the compositions of proteins orpeptides used in this method are recombinantly expressed type-specificor allele-specific proteins or type-specific peptides.

[0040] The present invention further relates to a kit for serotyping oneor more serological types or alleles of malaria parasite present in abiological sample, more particularly for detecting the antibodies tothese serological types of malaria parasites comprising:

[0041] at least one AMA1/E protein or AMA1/E peptide, as defined above,

[0042] a buffer or components necessary for producing the bufferenabling the binding reaction between these proteins or peptides and theanti-AMA1/E antibodies present in a biological sample,

[0043] means for detecting the immune complexes formed in the precedingbinding reaction,

[0044] possibly also an automated scanning and interpretation device fordetecting the presence of one or more serological types present from theobserved binding pattern.

[0045] The present invention also relates to the use of a peptide orprotein composition as defined above, for immobilization on a solidsupport and incorporation into a reversed phase hybridization assay,preferably for immobilization as parallel lines onto a solid supportsuch as a membrane strip, for determining the presence or the genotypeof malaria parasite according to a method as defined above. Combinationwith other type-specific or allele-specific antigens from other malariaparasites also lies within the scope of the present invention.

[0046] The present invention further relates to an AMA-1 specificantibody raised upon immunizing an animal with a peptide or proteincomposition of the present invention, with said antibody beingspecifically reactive with any of the polypeptides or peptides asdefined above, and with said antibody being preferably a monoclonalantibody.

[0047] The present invention also relates to an AMA1/E or AMA-1 specificantibody screened from a variable chain library in plasmids or phages orfrom a population of human B-cells by means of a process known in theart, with said antibody being reactive with any of the polypeptides orpeptides as defined above, and with said antibody being preferably amonoclonal antibody.

[0048] The AMA1/E specific monoclonal antibodies of the invention can beproduced by any hybridoma liable to be formed according to classicalmethods from splenic or lymph node cells of an animal, particularly froma mouse or rat, immunized against the Plasmodium polypeptides orpeptides according to the invention, as defined above on the one hand,and of cells of a myeloma cell line on the other hand, and to beselected by the ability of the hybridoma to produce the monoclonalantibodies recognizing the polypeptides which has been initially usedfor the immunization of the animals.

[0049] The antibodies involved in the invention can be labelled by anappropriate label of the enzymatic, fluorescent, or radioactive type.

[0050] The monoclonal antibodies according to this preferred embodimentof the invention may be humanized versions of mouse monoclonalantibodies made by means of recombinant DNA technology, departing fromparts of mouse and/or human genomic DNA sequences coding for H and Lchains from cDNA or genomic clones coding for H and L chains.

[0051] Alternatively the monoclonal antibodies according to thispreferred embodiment of the invention may be human monoclonalantibodies. These antibodies according to the present embodiment of theinvention can also be derived from human peripheral blood lymphocytes ofpatients infected with malaria, or vaccinated against malaria. Suchhuman monoclonal antibodies are prepared, for instance, by means ofhuman peripheral blood lymphocytes (PBL) repopulation of severe combinedimmune deficiency (SCID) mice, or by means of transgenic mice in whichhuman immunoglobulin genes have been used to replace the mouse genes.

[0052] The invention also relates to the use of the proteins or peptidesof the invention, for the selection of recombinant antibodies by theprocess of repertoire cloning.

[0053] Antibodies directed to peptides or single or specific proteinsderived from a certain strain may be used as a medicament, moreparticularly for incorporation into an immunoassay for the detection ofPlasmodium strains for detecting the presence of AMA-1 antigens, orantigens containing AMA-1, or AMA1/E epitopes, for prognosing/monitoringof malaria disease, or as therapeutic agents.

[0054] Alternatively, the present invention also relates to the use ofany of the above-specified AMA1/E monoclonal antibodies for thepreparation of an immunoassay kit for detecting the presence of AMA-1antigen or antigens containing AMA1/E epitopes in a biological sample,for the preparation of a kit for prognosing/monitoring of malariadisease or for the preparation of a malaria medicament.

[0055] The present invention also relates to a method for in vitrodiagnosis or detection of malaria antigen present in a biologicalsample, comprising at least the following steps:

[0056] (i) contacting said biological sample with any of the AMA1/Especific monoclonal antibodies as defined above, preferably in animmobilized form under appropriate conditions which allow the formationof an immune complex,

[0057] (ii) removing unbound components,

[0058] (iii) incubating the immune complexes formed with heterologousantibodies, which specifically bind to the antibodies present in thesample to be analyzed, with said heterologous antibodies conjugated to adetectable label under appropriate conditions,

[0059] (iv) detecting the presence of said immune complexes visually ormechanically (e.g. by means of densitometry, fluorimetry, colorimetry).

[0060] The present invention also relates to a kit for in vitrodiagnosis of a malaria antigen present in a biological sample,comprising:

[0061] at least one monoclonal antibody as defined above, with saidantibody being preferentially immobilized on a solid substrate,

[0062] a buffer or components necessary for producing the bufferenabling binding reaction between these antibodies and the malariaantigens present in the biological sample, and

[0063] a means for detecting the immune complexes formed in thepreceding binding reaction.

[0064] The kit can possibly also include an automated scanning andinterpretation device for inferring the malaria antigens present in thesample from the observed binding pattern.

[0065] Monoclonal antibodies according to the present invention aresuitable both as therapeutic and prophylactic agents for treating orpreventing malaria infection in susceptible malaria-infected subjects.Subjects include rodents such as mice or guinea pigs, monkeys, and othermammals, including humans.

[0066] In general, this will comprise administering a therapeutically orprophylactically effective amount of one or more monoclonal antibodiesof the present invention to a susceptible subject or one exhibitingmalaria infection. Any active form of the antibody can be administered,including Fab and F(ab′)₂ fragments. Antibodies of the present inventioncan be produced in any system, including insect cells, baculovirusexpression systems, chickens, rabbits, goats, cows, or plants such astomato, potato, banana or strawberry. Methods for the production ofantibodies in these systems are known to a person with ordinary skill inthe art. Preferably, the antibodies used are compatible with therecipient species such that the immune response to the MAbs does notresult in clearance of the MAbs before parasite can be controlled, andthe induced immune response to the MAbs in the subject does not induce“serum sickness” in the subject. Preferably, the MAbs administeredexhibit some secondary functions such as binding to Fc receptors of thesubject.

[0067] Treatment of individuals having malaria infection may comprisethe administration of a therapeutically effective amount of AMA1/Eantibodies of the present invention. The antibodies can be provided in akit as described below. The antibodies can be used or administered as amixture, for example in equal amounts, or individually, provided insequence, or administered all at once. In providing a patient withantibodies, or fragments thereof, capable of binding to AMA1/E , or anantibody capable of protecting against malaria in a recipient patient,the dosage of administered agent will vary depending upon such factorsas the patient's age, weight, height, sex, general medical condition,previous medical history, etc.

[0068] In general, it is desirable to provide the recipient with adosage of antibody which is in the range of from about 1 pg/kg-100pg/kg, 100 pg/kg-500 pg/kg, 500 pg/kg-, ng/kg, 1 ng/kg-100 ng/kg, 100ng/kg-500 ng/kg, 500 ng/kg-1 ug/kg, 1 ug/kg-100 ug/kg, 100 ug/kg-500ug/kg, 500 ug/kg-1 mg/kg, 1 mg/kg-50 mg/kg, 50 mg/kg-100 mg/kg, 100mg/kg-500 mg/kg, 500 mg/kg-i g/kg, 1 g/kg-5 g/kg, 5 g/kg-10 g/kg (bodyweight of recipient), although a lower or higher dosage may beadministered.

[0069] In a similar approach, another prophylactic use of the monoclonalantibodies of the present invention is the active immunization of apatient using an anti-idiotypic antibody raised against one of thepresent monoclonal antibodies. Immunization with an anti-idiotype whichmimics the structure of the epitope could elicit an active anti-AMA1/Eresponse (Linthicum, D. S. and Farid, N. R., Anti-Idiotypes, Receptors,and Molecular Mimicry (1988), pp 1-5 and 285-300)

[0070] Likewise, active immunization can be induced by administering oneor more antigenic and/or immunogenic epitopes as a component of asubunit vaccine. Vaccination could be performed orally or parenterallyin amounts sufficient to enable the recipient to generate protectiveantibodies against this biologically functional region, prophylacticallyor therapeutically. The host can be actively immunized with theantigenic/immunogenic peptide in pure form, a fragment of the peptide,or a modified form of the peptide. One or more amino acids, notcorresponding to the original protein sequence can be added to the aminoor carboxyl terminus of the original peptide, or truncated form ofpeptide. Such extra amino acids are useful for coupling the peptide toanother peptide, to a large carrier protein, or to a support. Aminoacids that are useful for these purposes include: tyrosine, lysine,glutamic acid, aspartic acid, cyteine and derivatives thereof.Alternative protein modification techniques may be used e.g.,NH₂-acetylation or COOH-terminal amidation, to provide additional meansfor coupling or fusing the peptide to another protein or peptidemolecule or to a support.

[0071] The antibodies capable of protecting against malaria are intendedto be provided to recipient subjects in an amount sufficient to effect areduction in the malaria infection symptoms. An amount is said to besufficient to “effect” the reduction of infection symptoms if thedosage, route of administration, etc. of the agent are sufficient toinfluence such a response. Responses to antibody administration can bemeasured by analysis of subject's vital signs.

[0072] The present invention more particularly relates to a compositioncomprising at least one of the above-specified peptides or a recombinantAMA1/E protein composition as defined above, for use as a vaccine forimmunizing a mammal, preferably humans, against malaria, comprisingadministering a sufficient amount of the composition possiblyaccompanied by pharmaceutically acceptable adjuvant(s), to produce animmune response.

[0073] Immunogenic compositions can be prepared according to methodsknown in the art. The present compositions comprise an immunogenicamount of a recombinant AMA1/E proteins or peptides as defined above,usually combined with a pharmaceutically acceptable carrier, preferablyfurther comprising an adjuvant.

[0074] The proteins of the present invention, preferably purified AMA1/Ederived from (3D7), are expected to provide a particularly usefulvaccine antigen, since the antigen is able to induce invasion inhibitoryantibodies as well as high titer antibodies that react withschizont-infected erythrocytes.

[0075] Pharmaceutically acceptable carriers include any carrier thatdoes not itself induce the production of antibodies harmful to theindividual receiving the composition. Suitable carriers are typicallylarge, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers; and inactive virus particles. Suchcarriers are well known to those of ordinary skill in the art.

[0076] Preferred adjuvants to enhance effectiveness of the compositioninclude, but are not limited to montanide, aluminum hydroxide (alum),N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) as found in U.S.Pat. No. No. 4,606,918, N-acetyl-normuramyl-L-alanyl-D-isoglutamine(nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE) and RIBI, which contains three components extracted frombacteria, monophosphoryl lipid A, trehalose dimycolate, and cell wallskeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. Any of the 3components MPL, TDM or CWS may also be used alone or combined 2 by 2.Additionally, adjuvants such as Stimulon (Cambridge Bioscience,Worcester, Mass.) or SAF-1 (Syntex) may be used. Further, CompleteFreund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA) may beused for non-human applications and research purposes. Other adjuvantsfor human research include ASO1, AS02A, AS02B, and AS03, AS04,AS05(GlaxoSmith Kline, Pa.), among others known or newly discovered.

[0077] The immunogenic compositions typically will containpharmaceutically acceptable vehicles, such as water, saline, glycerol,ethanol, etc. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, preservatives, and thelike, may be included in such vehicles.

[0078] Typically, the immunogenic compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid vehicles prior toinjection may also be prepared. The preparation also may be emulsifiedor encapsulated in liposomes for enhanced adjuvant effect. The AMA1/Eproteins of the invention may also be incorporated into ImmuneStimulating Complexes together with saponins, for example QuilA(ISCOMS).

[0079] Immunogenic compositions used as vaccines comprise a ‘sufficientamount’ or ‘an immunologically effective amount’ of the proteins of thepresent invention, as well as any other of the above mentionedcomponents, as needed. ‘Immunologically effective amount’, means thatthe administration of that amount to an individual, either in a singledose or as part of a series, is effective for treatment, as definedabove. This amount varies depending upon the health and physicalcondition of the individual to be treated, the taxonomic group ofindividual to be treated (e.g. nonhuman primate, primate, etc.), thecapacity of the individual's immune system to synthesize antibodies, thedegree of protection desired, the formulation of the vaccine, thetreating doctor's assessment of the medical situation, the strain ofmalaria infection, and other relevant factors. It is expected that theamount will fall in a relatively broad range that can be determinedthrough routine trials. Usually, the amount will vary from 0.01 to 1000ug/dose, more particularly from about 1.0 to 100 ug/dose most preferablyfrom about 10 to 50 ug/dose.

[0080] The proteins may also serve as vaccine carriers to presenthomologous (e.g. other malaria antigens, such as MSP-1₄₂ CSP, TRAP,LSA1, LSA3, Pfs25) or heterologous (non-malaria) antigens. In this use,the proteins of the invention provide an immunogenic carrier capable ofstimulating an immune response to other antigens. The antigen may beconjugated either by conventional chemical methods, or may be clonedinto the gene encoding AMA1/E fused to the 5′ end or the 3′ end of theAMA1/E gene. The vaccine may be administered in conjunction with otherimmunoregulatory agents.

[0081] The compounds of the present invention can be formulatedaccording to known methods to prepare pharmaceutically usefulcompositions, whereby these materials, or their functional derivatives,are combined in admixture with a pharmaceutically acceptable carriervehicle. Suitable vehicles and their formulation, inclusive of otherhuman proteins, e.g., human serum albumin, are described, for example,in Remington's Pharmaceutical Sciences (16th ed., Osol, A. ed., MackEaston Pa. (1980)). In order to form a pharmaceutically acceptablecomposition suitable for effective administration, such compositionswill contain an effective amount of the above-described compoundstogether with a suitable amount of carrier vehicle.

[0082] Additional pharmaceutical methods may be employed to control theduration of action. Control release preparations may be achieved throughthe use of polymers to complex or absorb the compounds. The controlleddelivery may be exercised by selecting appropriate macromolecules (forexample polyesters, polyamino acids, polyvinyl, pyrrolidone,ethylenevinylacetate, methylcellulose, carboxymethylcellulose, orprotamine sulfate) and the concentration of macromolecules as well asthe method of incorporation in order to control release. Anotherpossible method to control the duration of action by controlled releasepreparations is to incorporate the compounds of the present inventioninto particles of a polymeric material such as polyesters, polyaminoacids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.Alternatively, instead of incorporating these agents into polymericparticles, it is possible to entrap these materials in microcapsulesprepared, for example, interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsules andpoly(methylmethacylate)-microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(1980).

[0083] Administration of the compounds, whether antibodies or vaccines,disclosed herein may be carried out by any suitable means, includingparenteral injection (such as intraperitoneal, subcutaneous, orintramuscular injection), in ovo injection of birds, orally, or bytopical application of the antibodies (typically carried in apharmaceutical formulation) to an airway surface. Topical application ofthe antibodies to an airway surface can be carried out by intranasaladministration (e.g., by use of dropper, swab, or inhaler which depositsa pharmaceutical formulation intranasally). Topical application of theantibodies to an airway surface can also be carried out by inhalationadministration, such as by creating respirable particles of apharmaceutical formulation (including both solid particles and liquidparticles) containing the antibodies as an aerosol suspension, and thencausing the subject to inhale the respirable particles. Methods andapparatus for administering respirable particles of pharmaceuticalformulations are well known, and any conventional technique can beemployed. Oral administration may be in the form of an ingestable liquidor solid formulation.

[0084] The treatment may be given in a single dose schedule, orpreferably a multiple dose schedule in which a primary course oftreatment may be with 1-10 separate doses, followed by other doses givenat subsequent time intervals required to maintain and or reinforce theresponse, for example, at 1-4 months for a second dose, and if needed, asubsequent dose(s) after several months. Examples of suitable treatmentschedules include: (i) 0, 1 month and 6 months, (ii) 0, 7 days and 1month, (iii) 0 and 1 month, (iv) 0 and 6 months, or other schedulessufficient to elicit the desired responses expected to reduce diseasesymptoms, or reduce severity of disease.

[0085] The present invention also provides kits which are useful forcarrying out the present invention. The present kits comprise a firstcontainer means containing the vaccine or antibodies of the invention.The kit also comprises other container means containing solutionsnecessary or convenient for carrying out the invention. The containermeans can be made of glass, plastic or foil and can be a vial, bottle,pouch, tube, bag, etc. The kit may also contain written information,such as procedures for carrying out the present invention or analyticalinformation, such as the amount of reagent contained in the firstcontainer means. The container means may be in another container means,e.g. a box or a bag, along with the written information.

[0086] The contents of all cited references (including literaturereferences, issued patents, published patent applications, andco-pending patent applications) cited throughout this application arehereby expressly incorporated by reference.

[0087] Other features of the invention will become apparent in thecourse of the following descriptions of exemplary embodiments which aregiven for illustration of the invention and are not intended to belimiting thereof. The following MATERIALS AND METHODS were used in theexamples that follow.

[0088] Cloning and Expression:

[0089] A nucleotide construct encoding 449 amino acids of AMA1 of P.falciparum 3D7 clone, residue #83_(Gly)-531_(Glu) was commerciallysynthesized with an E. coli codon bias (Retrogen, San Diego, Calif.).The synthetic gene insert was ligated to the Nco I and Not I sites of amodified pET32 plasmid, called pWRMAL. The modifications in the plasmidinclude the replacement of the thioredoxin and other N-terminal tagswith sequences that resulted in minimal non-AMA1 amino acids fused tothe final recombinant protein and a tetr gene for tetracyclineresistance added. (Angov, E. et al.; unpublished). To correct a readingframe error the recombinant vector was cut with Nco I, end-filled withKlenow fragment and religated. The final construct contained 18 aminoacids fused to the N-terminus and 11 amino acids fused to theC-terminus. The resulting protein construct was designated as r-AMA1/E(MAHHHHHHPGGSGSGTMH (SEQ ID NO:3))-(AMA1 amino acids#83-531)-AAALEHHHHHH (SEQ ID NO:4)). The recombinant plasmid,pWRMAL-AMA1/E (FIG. 1) was transformed into E. coli Sure II cells andthe insert was sequenced on both strands. For protein expression therecombinant plasmid was transformed into a ‘redox modified’ host E. colistrain (Origami (DE3); Novagen, Madison, Wis.). Origami (DE3) cells aretetracycline and kanamycin resistant. Expression of r-AMA1/E protein wasconfirmed by IPTG induction in shake flask cultures and glycerol stockswere prepared.

[0090] Fermentation (GMP Production):

[0091] Expression of r-AMA1/E protein was carried out in a 10 Lbioreactor (New Brunswick Scientific, Edison, N.J.) at the lab scale andin a 300 L bioreactor (New Brunswick Scientific, Edison, N.J.) at theWalter Reed Army Institute of Research Pilot Bioproduction Facility.Medium consisting of Super Broth containing 0.8% glycerol and 12.5 μgml⁻¹ tetracycline was inoculated with a 3 L overnight culture startedfrom a Production Seed Lot #0788. The bioreactor temperature wasmaintained at 27° C.; pH 7.2 and agitation at 400 rpm. At a cell densityOD₆₀₀=7.0, IPTG was added, to a final concentration of 0.1 mM. One hourlater cells were harvested by centrifugation and frozen at −80° C.Aliquots (10 to 150 g) from this production were used to develop apurification and refolding process of r-AMA1/E protein at the lab priorto scale-up (1500 g) in a GMP environment.

[0092] Plasmid Stability:

[0093] The presence of recombinant plasmid in E. coli Origami (DE3)cells after fermentation was determined by plating an appropriatedilution of cells on LB agar plates containing either tetracycline (12.5μg ml⁻¹) or ampicillin (100 μg ml⁻¹) (selective plates) and on LB agarplates containing no antibiotic (non-selective plates). The percentplasmid retention [No. colonies on selective plates/No. colonies onnon-selective plates] was calculated using colony counts on appropriatedilution plates containing between 30 and 300 colonies.

[0094] Metal Affinity Purification (Lab-Scale):

[0095] All buffers were endotoxin free and maintained chilled; allchemicals used during purification were ACS certified or the next bestavailable grade. Purification was carried out at room temperature on aWaters-600 liquid chromatography system configured to runPharmacia-Amersham HR columns. Cell paste was thawed and suspended in 5times w/v of buffer-A (15 mM Na₂HPO₄, 5.1 mM KH₂PO₄, 450 mM NaCl; pH7.4) and mixed until homogenous. A solution of 20% sodium N-lauroylsarcosine (sarkosyl) was added to a final concentration of 5% detergent.This suspension was mixed and the E. coli cells were disrupted byhigh-pressure microfluidization (Model 1109, Microfluidic Corp., Newton,Mass.). The cell lysate was cleared by centrifugation at 22,000× g andthe supernatant was diluted 4 fold in buffer-A before loading onto aNi⁺²-NTA Superflow column (Qiagen, Valencia, Calif.; 0.5 ml packed resinper gram paste). The Ni⁺² column was pre-equilibrated with buffer-B(buffer-A containing 1.25% sarkosyl; pH 7.4). After loading the lysate,the Ni⁺² column was washed with 20 column volumes (CV) of the buffer-C(buffer-A with 10 mM imidazole, 0.125% sarkosyl; pH 7.4) followed by 20CV of buffer-D (20 mM sodium phosphate, 25 mM imidazole, 0.125%sarkosyl; pH 8.0). Bound proteins were eluted from the column inbuffer-D containing 500 mM imidazole (pH 8.0).

[0096] Refolding:

[0097] The Ni⁺² elution was diluted 40 fold (v/v), rapidly, in degassedbuffer-E (20 mM sodium phosphate, 1 mM EDTA, 1 mM reduced glutathione(GSH), 0.25 mM oxidized glutathione (GSSG); pH 8.0). The refoldingbuffer was prepared fresh and refolding was carried out at roomtemperature (˜22° C.) for a minimum of 15 h under nitrogen. The finalprotein product, resulting from this refolding protocol, was referred toas AMA1/E . Several other variations to the above refolding protocolwere also tested. One such variation included reduction of the Ni⁺²eluted proteins with 5 mM DTT for 1 h at 37° C. before refolding. Theprotein after reduction and refolding followed by ion-exchangepurification was referred to as RR-AMA1/E.

[0098] Ion-Exchange Purification:

[0099] Ion-exchange column resins were sanitized with 0.2 N NaOH beforeuse and then equilibrated to initial binding conditions. After therefolding step, AMA1/E protein was concentrated on a DEAE Sepharoseanion-exchange column (Amersham Pharmacia Biotech, Piscataway, N.J.);0.25 ml packed resin per gram paste), the column was pre-equilibratedwith buffer-E without the GSH/GSSG. After loading the protein, thecolumn was washed with a minimum of 30 CV of the same equilibrationbuffer followed by 10 CV of buffer-F (5 mM sodium phosphate, 50 mM NaCl,1 mM EDTA; pH 8.0). AMA1/E was eluted in buffer-F containing a finalconcentration of 100 mM NaCl (pH 8.0). AMA1/E eluted from the DEAEcolumn was pH adjusted to 5.7 by the addition of 1M NaH₂PO₄.H₂O andloaded on a SP Sepharose cation-exchange column (Amersham PharmaciaBiotech; 0.15 ml packed resin per gram paste), pre-equilibrated withbuffer-G (50 mM sodium phosphate, 0.1 mM EDTA, 100 mM NaCl; pH 5.7). Thecolumn was washed with 20 CV of buffer-G containing a final 275 mM NaCl(pH 5.7), followed by 10 CV of a pH exchange buffer (5 mM sodiumphosphate, 0.1 mM EDTA; pH 7.1). AMA1/E was eluted from the column informulation buffer (23.5 mM NaH₂PO₄.H₂O, 37.5 mM NaCl, 0.1 mM EDTA; pH7.1).

[0100] Formulation, Lyophilization and Storage:

[0101] Purified AMA1/E protein eluted from the SP column was quantifiedby Bio-Rad DC protein assay (BioRad, Richmond, Calif.). AMA1/E wasvialed at 100 μg ml⁻¹, 65 μg protein per vial, in the final formulationbuffer (23.5 mM NaH₂PO₄.H₂O, 30 mM NaCl, 0.1 mM EDTA, 3.15% sucrose; pH7.1) and lyophilized.

[0102] Residual Sarkosyl and Endotoxin Content Determination:

[0103] The residual sarkosyl in purified AMA1/E protein preparations wasmeasured by a reversed-phase HPLC method (Burgess, R. R., 1996, Meth.Enzymol. 273, 145-149). Endotoxin content was estimated using thechromogenic Limulus Amebocyte Lysate (LAL) endpoint assay (Associates ofCape Cod, Falmouth, Mass.). Dilutions of all protein samples and LALstandard were prepared in pyrogen-free vials. Positive control solutionsprepared for the standard curves ranged from 1 endotoxin unit (EU) ml⁻¹to 0.06 EU ml⁻¹, in two-fold serial dilutions. A 96-well plate heaterwas used for incubation at 37° C. for 20 min and the assay was carriedout as per the manufacturer's instruction. The plates were read at 405nm on V_(max) kinetic microplate reader (Molecular Devices Corp.,Sunnyvale, Calif.).

[0104] Purity and Stability Analysis:

[0105] AMA1/E was evaluated for purity on precast polyacrylamide gels(4-12% gradient Bis-Tris, Invitrogen, Carlsbad, Calif.), run underreduced and non-reduced conditions, with 5-10 μg protein loaded perwell. Gels were stained with Coomassie blue, destained, scanned on aLaser densitometer and acquired data was analyzed by ImageQuant 5.1software (Molecular Dynamics, Sunnyvale, Calif.). Residual host cellprotein (HCP) content, was assessed by ELISA and Western blotting, usingcommercially available kits (Cygnus Technologies, Plainville, Mass.).The HCP standard recommended by the manufacturer was used. In additionto this control, a lysate of the host E. coli Origami (DE3) (expressinga P. vivax MSP1 protein construct) was also tested as a standard between1000 and 15 ng ml⁻¹, protein concentration, to determine if the kit wascapable of detecting proteins from this specific host E. coli. The totalprotein in the Origami (DE3) lysate was estimated by BCA protein assay(Pierce). HCP ELISA was performed twice, using concentrations of AMA1/E,between 10,000 and 80 ng ml⁻¹, as per the ‘standard procedure’recommended by the manufacturer. Immunoblotting for HCP determination(Cygnus Technologies kit) was carried out using the HCP standardprovided by the manufacturer and also using the Origami (DE3) E. colilysate, between 4000 and 250 ng protein per well run on a reducing gel.The proteins were electrophoretically transferred to a nitrocellulosemembrane and the western blot assay was performed as per themanufacturer's instructions. Stability of AMA1/E was determined bySDS-PAGE and western blotting of protein samples drawn monthly fromaliquots stored at −80° C., −30° C., 4° C., 22° C. (RT) and 37° C.

[0106] Primary Structure Analysis:

[0107] Purified AMA1/E protein was sequenced by automated Edman'sdegradation method on an Applied Biosystems model 477A proteinsequencer, in-line with a HPLC (Applied Biosystems model 120A), fordetection of phenylthiohydantoin-derived amino acids. Protein sampleswere analyzed by, Matrix Assisted Laser Desorption Ionization-Time offlight mass spectrometer (MALDI-TOF; Voyager Biospectrometry RP system,Applied Biosystems), using Sinapinic acid matrix. Lysozyme andCytochrome C were used as mass standards.

[0108] Reduction, Alkylation and Free Thiol Analysis:

[0109] AMA1/E protein was reduced with a 100-fold molar excess of DTTover cysteines in presence of either 4 M urea (for SDS-PAGE) or 4 Mguanidine-HCl (for RP-HPLC and Ellman's test) at 50° C. for 1 h.Alkylation was carried out in presence of either 4 M urea or 4 Mguanidine-HCl, along with 1000-fold molar excess of iodoacetamide overcysteines, for 1 h at room temperature in the dark. Free sulfhydrylgroups were estimated in the presence and absence of 4 M guanidine-HClby Ellman's reagent (5,5′-dithio-bis-3-nitrobenzoic acid) (Ellman, G.L., 1959, Arch. Biochem. Biophys. 82, 50-77). L-cysteine was used toplot the standard curve.

[0110] Gel-Permeation (GPC) and Reversed-Phase (RPC) Chromatography:

[0111] HPLC analysis of purified protein was carried out using aWaters-510 HPLC pump, connected to Waters-712 WISP autosampler andcontrolled by Millenium Release 3.2 chromatographic software (WatersCorp., Milford, Mass.). Waters-996 PDA detector was used to monitor theelution profiles. For GPC analysis a Shodex Protein KW-803 column(Waters Corp., Milford, Mass.) was used with 10 μg protein injection.Buffer system consisted of 20 mM sodium phosphate, 100 mM K₂SO₄ (pH7.15) at 0.5 ml min⁻¹ flow rate. The column was calibrated withmolecular weight standards (BioRad). RPC analysis was done with a C8Aquapore RP-300 Å column, 7μ, 30_(—)2.1 mm (P E Brownlee, Norwalk,Conn.) at 0.5 ml min⁻¹ flow-rate and 4-12 μg protein per load. SolventA: 0.05% trifluroacetic acid (TFA) in H₂O; solvent B: 0.05% TFA inacetonitrile. The solvent gradient consisted of 100% solvent A for 5min, 100% to 30% solvent A over 15 min, 30% to 0% solvent A over 5 minand back to 100% solvent A over 5 min.

[0112] Immune Reagents:

[0113] Rat monoclonal antibody, 4G2dc1 (used at 1.5 μg ml⁻¹ onimmunoblots and ELISA), recognizes a disulphide bond dependentconformational epitope on P. falciparum AMA1 (Kocken, et al., 1998,supra), was kindly provided by Dr. Alan W. Thomas, Biomedical PrimateResearch Center, Rijswijk, The Netherlands. A pool of immune human sera(used at 1:1000 dilution on immunoblots) was collected from an endemicarea in Western Kenya; the same dilution of a pool of commerciallyobtained normal human serum (The Binding Site Limited, Birmingham, UK)was used as a negative control.

[0114] Immunoblotting:

[0115] Proteins were separated on SDS-PAGE and electrophoreticallytransferred to a nitrocellulose membrane (Towbin et al., 1979,Biotechnology 24, 145-149). The blot was blocked with 0.5% casein and0.3% Tween-20 containing phosphate buffered saline (PBS). Appropriatedilution of primary antibody in PBST (PBS with 0.05% Tween-20) wasincubated for 2 h. The blot was washed with PBST and then incubated with1:5000 dilution of HRP conjugated secondary antibody (SouthernBiotechnology Associates, Birmingham, Ala.) for 1 h. After washing withPBST, the blot was developed either with Super Signal Chemiluminescentsubstrate (Pierce, Rockford, Ill.) or with BM Blue POD substrate (Roche,Indianapolis, Ind.) according to the manufacturer's recommendation.

[0116] Indirect-Immumofluorescence Assay (IFA):

[0117] Recognition of P. falciparum 3D7 schizonts, by anti-ANA1antibodies was tested by IFA. Thin blood smears were fixed with chilledmethanol and serial dilutions of sera in PBST were incubated for 2 h.Slides were washed three times with PBST and incubated with a 1:100dilution of goat anti-rabbit IgG FITC-labeled antibodies (SouthernBiotechnologies Associates) for 1 h. Slides were washed, anti-fadesolution (Molecular Probes, Inc, Eugene, Oreg.) was applied and read ona UV fluorescence microscope. IFA titers were determined as the lastserum dilution with a positive recognition of the parasite compared tothe negative adjuvant control rabbit serum diluted 1:20. The assay wasdone twice on separate days.

[0118] Rabbit Immunization and Total IgG Purification:

[0119] Groups of three NZW rabbits were immunized, three times with 100μg of lab-grade refolded AMA1/E (animal codes R-1, 2, 3); reduced andrefolded protein RR-AMA1/E (R-4, 5, 6) or its reduced and alkylated formRA-AMA1/E (R-7, 8, 10). A group of 3 rabbits received 50 μg (V-2, 3, 4)or 100 μg (V-9, 10, 11) of AMA1/E protein produced under GMPenvironment. A control group of 3 rabbits (R-9, V45, 45) were given PBSalong with the adjuvant. Formulation was prepared by adding 70% (v/v)Montanide® ISA-720 (Seppic Inc. Paris, France) to 30% antigen to make atotal 1 ml emulsion per dose. The immunization was given subcutaneous atmultiple sites, with a three wk interval between consecutiveimmunizations. Serum samples were collected 2 wk after eachimmunization. Rabbits were bled out 2 wk after the last immunization.Total IgG was purified from 9 ml pooled rabbit sera (lab-grade AMA1/Eand RA-AMA1/E group). The adjuvant control IgG was purified from asingle animal (R9). IgG purification was done on a 5 ml protein-GSepharose column (Amersham Pharmacia Biotech. Piscataway, N.J.) usingIgG binding and elution buffers (Pierce, Rockford, Ill.), according tothe manufacturer's recommendation.

[0120] ELISA:

[0121] Antibody response was evaluated by enzyme-linked immunosorbentassay (ELISA). Ninety six well microtiter plates (Dynax, Chantilly, Va.)were coated with 100 ng per well of either RA-AMA1/E or AMA1/E ,incubated overnight at 4° C., plates were blocked for 1 h with PBSTcontaining 5% casein (Sigma, St. Louis, Mo.) and washed with PBST.Consecutive dilutions of individual rabbit sera were incubated for 2 hat room temperature. Plates were washed and 1:4000 dilutedHRP-conjugated secondary antibody was incubated for 1 h. Plates werewashed and developed for 25 min with ABTS-peroxidase substrate(Kirkegaard & Perry Laboratories, Gaithersburg, Md.). OD₄₀₅ was recordedand comparative ELISA titers were calculated using regression analysison the titration curve. The ELISA was repeated 3 times for eachindividual serum, in triplicate wells, on separate days. CompetitiveELISA was carried out using sera from 3 rabbits immunized with lab-gradeAMA1/E and two rabbits in the RA-AMA1/E group. The sera were diluted1:1000 and pre-incubated in solution with 15 μg ml⁻¹ of either AMA1/E orPA-AMA1/E or with BSA, overnight at 4° C. on a shaker. The tubes werecentrifuged at 15,000 rpm for 15 min and the supernatants were analyzedby ELISA (as described above), with AMA1/E coated on plates. Thecompetition assay was done 3 times.

[0122] Parasite Culture and Growth Inhibition Assay (GIA):

[0123]P. falciparum clone 3D7 cultures were prepared as describedpreviously (Haynes et al., 2002, Erythrocytic Malaria Growth or InvasionInhibition Assays (GIA) with Emphasis on Suspension Culture GIA, Chapter51, in Malaria Methods and Protocols, ed. Denise L. Doolan, Methods inMolecular Medicine, The Humana Press Inc., NJ), in 48-well plates, keptin suspension cultures angled on a rotator platform or under staticconditions. All cultures contained a final 10% heat inactivated normalhuman serum in bicarbonate-containing RPMI 1640. Plates were gassed with5% O₂, 5% CO₂ and heat-sealed in plastic bags. Synchronized culturesadjusted to 0.2% late ring stages were mixed with test or control serumor IgG (dialyzed into RPMI-NaOH) to a final hematocrit of 4%. In orderto assess the antigen specificity of the antibody mediated inhibitions,antigens (AMA1/E or RA-AMA1/E ) were added to the IgG preparationsbefore testing in the GIA. The final concentration of antigens in theGIA was 5.3 μg ml⁻¹ (limited by low solubility of RA-AMA1/E protein).Merozoites were released after approximately 34 h and developing ringstages were harvested 14 h post invasion, stained with Hoechst dye-33342and analyzed by flow-cytometry (Haynes et al., 2002, supra). Thefluorescence signal was determined for a minimum of 40,000 erythrocytesgated on forward scatter. The fluorescent signal of ring-infectederythrocytes was about 20 times that of uninfected erythrocytes andschizont-infected erythrocytes, if present, had another 20-fold increasein signal. Almost all (>99%) of the parasites harvested from the assayswere ring forms or early trophozoite stages, as confirmed by spot checksof Giemsa-stained thin smears. Percentage inhibition was calculated fromthe mean parasitaemia of triplicate test and control wells as100%—(test/control). Sera from rabbits immunized with the adjuvant andPBS were used as controls in the GIA. Prebleeds from individual rabbitswas also tested.

[0124] Statistical Analysis:

[0125] Microsoft Excel was used to calculate the p values for 2-tailed ttests and the correlation coefficients (R²).

EXAMPLE 1

[0126] Fermentation of E. coli Origami (DE3) Expressing the r-AMA1/EProtein at 10L and 300L Scale:

[0127] The synthetic gene cloned in the vector pWRMAL was sequenced andthe translation of this gene sequence revealed no amino acid changesfrom the published 3D7 clone sequence (GenBank™ Accession No U65407.1).Fermentation conditions were developed in a 10 L bioreactor and laterscaled-up to a 300 L GMP fermentation. The 10 L fermentation routinelyresulted in 150 gm cell paste while the 300 L fermentation resulted in4.5 kg cell paste. The final plasmid stability for the GMP fermentationwas 36%. Although the use of Origami (DE3) increased the proportion ofr-AMA1/E in the soluble fraction (compared to the conventional BL21(DE3) strain), protein fractionation experiments showed that a majorityof r-AMA1/E was still localized in the insoluble fraction (data notshown).

EXAMPLE 2

[0128] Extraction of r-AMA1/E in Sarkosyl and Its Enrichment by Ni⁺²Affinity Chromatography:

[0129] Aliquots were taken from the GMP cell paste lot and a scalablerefolding and purification process was developed. During cell lysissoluble and insoluble forms of r-AMA1/E were extracted with buffercontaining 5% sarkosyl. The r-AMA1/E constituted ˜1-2% of total cellprotein estimated by laser densitometry of a SDS-PAGE run under reducedconditions (FIG. 2A, lane 1). Following the first step of purificationover Ni⁺²column, r-AMA1/E was enriched to ˜40% of total protein (FIG.2A, lane 2). A large fraction of r-AMA/E present in the Ni⁺² elution,was aggregated as seen on a non-reduced SDS-PAGE (data not shown).

EXAMPLE 3

[0130] Optimization of the Refolding Conditions:

[0131] In order to find the optimal refolding conditions, the Ni⁺²elution was subjected to rapid dilution in refolding buffers of varyingGSH/GSSG ratios, at pH 8.0, in phosphate buffer. Serial dilutions ofthese test refolding mixtures were coated on a microtiter plate andELISA reactivity against the conformation specific, inhibitory,monoclonal antibody 4G2dc1, was used as a measure of folding efficiency;while the reactivity to a monoclonal anti-hexa-histidine antibody wasused to confirm equivalent coating efficiency. Ratios of GSH/GSSG testedfor refolding included 1/0.1 mM, 1/0.25 mM, 1/1 mM, and 0.1/1 mMrespectively, while phosphate buffer containing EDTA (pH 8.0) alone wasused as a control. The GSH/GSSG ratios of 1/0.1 mM and 1/0.25 mM werefound to be equally efficient, both of which gave 4G2dc1 reactivityabout 5 times higher than the phosphate buffer control. As the GSH/GSSGratio of 1/0.25 mM had been previously reported for efficient refoldingof P. chabaudi AMA1 and more recently, the same was used to refold P.falciparum AMA1 expressed in E. coli (Crewther et al., 1996, Infect.Immun. 64, 3310-3317; Hodder et al., 2001, supra), we chose this ratioto refold r-AMA1/E . After refolding r-AMA1/E was designated as AMA1/E .A minimum 40-fold dilution of the Ni⁺² elution to about 20 μg ml⁻¹protein during refolding, was found necessary to minimize aggregation.No significant increase in monomer yield of AMA1/E was found if the Ni⁺²elution was first reduced with DTT prior to refolding, and therefore,the GMP purification process was carried out using the Ni⁺² elutionwithout reduction. The presence of low concentrations of sarkosyl(0.003%) in the refolding mix eliminated the need for a cosolvent duringrefolding.

EXAMPLE 4

[0132] Ion-Exchange Chromatography Was Used to Purify AMA1/E toHomogeneity:

[0133] After 15 h incubation in the refolding buffer, AMA1/E wasconcentrated on a DEAE anion-exchange column and its monomeric form waseluted with 100 mM NaCl, while the impurities and AMA1/E aggregatesremained bound to the column. The percent purity of AMA1/E after thisstep was ˜90% of the total protein eluted (FIG. 2A, lane 3). The pHadjustment step from pH 8.0 to 5.7 was needed to bind the majority ofAMA1/E to the SP cation-exchange column. This pH change had no effect onthe solubility of AMA1/E or its reactivity to immune reagents. AMA1/Ebound to the cation-exchanger was eluted with the final formulationbuffer, eliminating the need for an additional buffer exchange stepbefore formulation. The final yield of AMA1/E was about 0.75 to 1 mg L-1culture with >99% purity estimated by laser densitometry of Coomassieblue stained gels (FIG. 2A, lane 4). RR-AMA1/E also gave similar yieldand purity (data not shown).

EXAMPLE 5

[0134] Lyophilized Formulation of AMA1/E Along with Sucrose and EDTA WasStable:

[0135] A final 3.15% sucrose excipiant was added for stabilization andcake formation during lyophilization. AMA1/E, vialed at 100 μg ml⁻¹ in0.65 ml aliquots, was found to be stable in its lyophilized form at 37°C., 22° C. and 4° C. over a 24 wk period, with no signs of breakdown oraggregation. Solution or lyophilized forms of AMA1/E stored at −30° C.or −70° C. showed equivalent stability (data not shown).

EXAMPLE 6

[0136] Formulated AMA1/E Product Has Very Low Residual Endotoxin, HostCell Proteins or Sarkosyl Content:

[0137] The endotoxin content of purified AMA1/E under lab conditions wasbetween 3 to 5 EU's per 50 μg protein but dropped to below 0.06 EU(lowest value detectable by LAL assay) per 50 μg protein in GMPpurification. No residual sarkosyl was detected with an RP-HPLC basedassay (minimum detection limit 0.0005%). The HCP content was determinedby ELISA, using an anti-E. coli antibody kit, capable of quantitativelydetecting 15 ng ml⁻¹ HCP using the Origami (DE3) E. coli lysate (thelowest concentration of HCP tested). AMA1/E sample at 10,000 ng m⁻¹showed 54 and 44 ng ml⁻¹ HCP (in two tests), giving the final purity of99.4%. Purity of AMA1/E was also tested by western blot HCPdetermination kit (Cygnus). The Origami (DE3) lysate was used aspositive control at 4000 to 250 ng protein per well (FIG. 2B). All thepositive bands at 4000 ng per well (FIG. 2B, lane 1) were also observedat 1000 ng per well (FIG. 2B, lane 3). Below 1000 ng per well, many E.coli protein bands were not detectable. No E. coli specific bands wereseen in AMA1/E lanes with up to 2 μg AMA1/E loaded per well (FIG. 2B,lane 11).

EXAMPLE 7

[0138] AMA1/E Had the Predicted Primary and Tertiary Structure with NoFree Cysteines:

[0139] The primary sequence analysis of AMA1/E identified the first 24N-terminal amino acids to be ahhhhhhpggsgsgtmhGAEPAP (SEQ ID NO:5) (P.falciparum AMA1 specific residues in capital). The methionine at theN-terminal could not be identified. The MALDI-TOF mass spectrometeranalysis showed an average mass at 54,656 Da, while the predicted massof AMA1/E was 54,633 Da. The final product was evaluated for homogeneityand the presence of multimers by analytical RPC and GPC. A single peakwas seen on both GPC and RPC elution profiles, giving evidence of ahomogenous product (FIG. 3A, B). The RPC elution profile of AMA1/E ,shifted towards higher hydrophobicity under reducing condition (FIG. 3B,broken line). This indicates exposure of the protein hydrophobic core,upon DTT reduction, which otherwise, remained buried due to compactfolded state, stabilized by disulphide bond formation.

[0140] The primary structure of AMA1/E is expected to contain 16cysteine residues, the presence of any free cysteines in the finalproduct, which would have indicated incorrect folding. The free cysteinecontent was determined by Ellman's test. Ellman's analysis was alsocarried out in the presence of 4M GuHCl to unmask any sulfhydryl groupsburied in the hydrophobic core of the protein. Ellman's test detected nofree sulfhydryl groups in up to 5 μM AMA1/E , both in the presence andabsence of 4M GuHCl (minimum detection limit 0.1 μM free sulfhydryl).The absence of free cysteines was further confirmed by treating AMA1/Ewith an alkylating agent before and after reduction. Mobility of AMA1/Eon non-reduced SDS-PAGE showed no observable change after treatment withiodoacetamide (FIG. 4A, lanes 1 and 2), while its reductive-alkylationcaused significant decrease in mobility (FIG. 4A, lane 3). A recombinantP. vivax MSP-1 p42 fragment (Dutta et al., 2001, Infect. Immun. 69,5464-5470), which was predicted to contain a single free cysteine, wasused as a positive control in both the Ellman's and alkylation analysisand this free cysteine was identified in both tests (data not shown).The above tertiary structure analysis also suggests that, as in the caseof P. chabaudi AMA1 (Hodder et al., 1996, supra), the majority of AMA1/Emolecules also had all 16 cysteines cross-linked by disulphide bonds.

EXAMPLE 8

[0141] AMA1/E Reacts with Conformation Dependent Immune Reagents:

[0142] AMA1/E reacted with the monoclonal antibody 4G2dc1 on immunoblot(FIG. 4B, lane 1). This monoclonal antibody recognizes a reductionsensitive epitope on AMA1 of P. falciparum (Kocken et al., 1998 supra).Reactivity on immunoblot was also observed with a hyper-immune malariaendemic serum pool from Kenya (FIG. 4C, lane 1). Alkylation of AMA1/Ecaused no change in its reactivity to the above two immune reagents(FIG. 4B, lane 2 and 3C, lane 2). However, significant loss ofreactivity to both immune reagents was observed uponreductive-alkylation (FIG. 4B, lane 3 and 3C, lane 3), furtherconfirming the presence of critical reduction sensitive epitopes onAMA1/E .

EXAMPLE 9

[0143] AMA1/E Was Found to Be Immunogenic in Rabbits:

[0144] Immunization of rabbits, with lab-grade AMA1/E and RR-AMA1/E at100 μg per dose was done to determine if one form was immunologicallysuperior to the other. A group of 3 rabbits was immunized with 100 μgper dose RA-AMA1/E to determine if disulphide bond independent epitopesalso contributed towards the induction of inhibitory anti-AMA1antibodies. The AMA1/E protein produced under GMP conditions wasimmunized at 50 and 100 μg per dose to determine the immunogenicity atthe two doses (50 μg is the expected human dose). No apparent signs oftoxicity of the antigen-adjuvant combination was observed in theimmunized animals. Table 1 shows the mean log ELISA titer of immunizedgroups with either AMA1/E or RA-AMA1/E coated on plates. Rabbits in thelab-grade AMA1/E group (R-1, 2, 3) showed high titer antibodies againstAMA1/E protein. No significant difference in the titer was observedbetween 50 and 100 μg GMP protein immunized groups (data not shown),hence all six rabbits are represented by a single group in Table 1. TheAMA1/E specific titers observed in the 50 and 100 μg GMP produced AMA1/Egroup (V-2, 3, 4, 9, 10, 11) were higher than the 100 μg lab-gradeAMA1/E group (2 tailed t test, p=2.5E-04), The RR-AMA1/E group (R-4, 5,6) also had high ELISA titer against AMA1/E coated wells. The mean titerin the RR-AMA1/E group was slightly lower than the lab-grade AMA1/Eprotein immunized group although the difference was not statisticallysignificant (p=8.2E-01). The ELISA titers against the RA-AMA1/E proteincoated wells for lab-grade, GMP produced AMA1/E and RR-AMA1/E groupswere lower as compared to the refolded AMA1/E coated wells (p=2.5E-02,1.2E-04 and 1.8E-02 respectively). One of the 3 rabbits (R-8) immunizedwith RA-AMA1/E died while handling. Although, the two remaining RA-AMA1/E immunized rabbits (R-7, 10) had high titer of antibody againstRA-AMA1/E coated wells, the titer was lower against the refolded AMA1/Ecoated wells. This difference was not statistically significant(p=3.8E-01). TABLE 1 Mean Log Number of Mean Log ELISA titer IFA titerMean GIA data animals AMA1/E RA-AMA1/E Positive percent Immunization pergroup coated coated N Schizonts N inhibition N Lab-grade 3 5.60 4.94 34.41 2 57 (17) 3 AMA1/E, 100 ug (0.04) (0.20) (0.35) 68 (11)* 3* GMPAMAl/E, 6 6.01 5.29 3 4.71 1 84 (4) 2 50 & 100 ug (0.13) (0.22) (0.25)RR-AMA1/E 3 5.57 5.01 3 4.06 2 29 (22) 2 100 ug (0.19) (0.17) (0.35) 41(25)* 2* RA-AMA1/E 2 4.83 5.01 3 2.43 2 5 (1) 1 100 ug (0.15) (0.17)(0.11) Adjuvant 3 0.00 0.00 2 0.00 1 −2 (2) 2 # The percent-inhibitionof parasite growth, in a 1-cycle assay, under suspension conditions, at1:5 serum dilution are shown. Percentage inhibition was calculated fromthe mean parasitaemia of triplicate test and control wells as 100% −((test/control) × 100). Inhibitions in GIA were relative to adjuvantcontrol sera or culture media alone. Initial parasitemias were 0.2% andthe final # control parasitemias ranged from 2.2% to 3.6%. Compared withgrowth in media alone (100%), there were no significant differences inthe final parasitemia of 12 pre-immune sera (mean ± SD = 97%, ± 4%suspension, 102% ± 1% static) or 3 adjuvant only sera (99% ± 3%suspension, 106% ± 2% static).

[0145] Sera from all AMA1/E immunized rabbits tested positive by IFAwith late stage schizonts of 3D7 parasites (FIG. 5A). Table 1 shows themean log IFA titer of the groups. The IFA titer in the GMP producedAMA1/E group was higher than the lab-grade AMA1/E group, although thedifference was not significant (p =2.7E-01). The lab-grade AMA1/E grouphad slightly higher IFA titers than the RR-AMA1/E group, this differencewas also not significant (p=2.8E-01). The IFA titer of lab-grade, GMPproduced AMA1/E and the RR-AMA1/E groups, were significantly higher thanthe RA-AMA1/E group titers (p 5.4E-03, 1.5E-05 and 8.6E-03respectively).

[0146] Rabbit antibodies reacted with two bands one 76 kDa and anotherat ˜62 kDa on western blot of an SDS extract of schizont richpreparation of P. falciparum 3D7 parasites (FIG. 5B, lane 1). Thesebands most likely correspond to the previously reported 83 and 66 kDafull length and processed forms of AMA1 in P. falciparum (Narum andThomas, 1994, supra; Howell et al., 2001, supra); difference in apparentmolecular weights observed here may be a result of difference in PAGEconditions.

EXAMPLE 10

[0147] Anti-AMA1/E Antibodies Inhibit in vitro Growth of the Parasite:

[0148] The growth inhibition assay (GIA) of homologous 3D7 P. falciparumparasites was carried out with sera obtained from the immunized rabbits.Table 1 shows the mean percent inhibition, under suspension conditions,at 1:5 dilution obtained for each of the immunized groups. The lab-gradeand GMP produced AMA1/E group sera showed significant inhibition ofparasite growth, compared to the adjuvant controls (p=2.4E-02, 1.1E-09respectively). The lab-grade AMA1/E group sera analyzed under static GIAconditions, gave even higher inhibition compared to the suspensionculture, although the difference between static and suspension culturevalues was not significant (p=8.4E-02) (Table 1). Rabbits immunized with50 and 100 μg doses showed no significant difference in the percentinhibition (data not shown). The RR-AMA1/E immunized group sera showedlower level of inhibition when compared lab-grade AMA1/E group, bothunder suspension (p value=1.5E-01) and static conditions, (p=1.9E-02);the difference was not statistically significant under suspensionconditions. There was a positive correlation between log ELISA and logIFA titers (R²=0.84). There was also a positive correlation between logELISA against the AMA1/E protein coated wells and the percent GIA(R²=0.81). A positive correlation was also observed between the log IFAtiter and percent inhibition (R²=0.77). The above R² values werecalculated using data from all the immunized rabbits in all the groups.No inhibition was seen in RA-AMA1/E group compared to the lab-gradeAMA1/E group (p=3.1E-02). In comparison to the growth in media alone(100%) there were no significant differences in the final parasitaemiaof 12 pre-immune sera (mean ±SD=97±4%) or the 3 adjuvant alone sera(99±3%). Whole serum from one of the rabbits (R-3), which showed 44%inhibition in the one cycle suspension GIA, was used in a two-cyclesuspension GIA at the same dilution. Inhibition of 87% was seen,indicative of cumulative inhibition over two cycles.

[0149] Inhibition of parasite growth was also observed with IgG purifiedfrom pooled sera of the rabbits within the lab-grade AMA1/E, RA-AMA1/Eand the adjuvant control (FIG. 6). The percent inhibition in the AMA1/Egroup was significantly higher than the adjuvant control at 0.18, 0.35and 0.7 mg ml⁻¹ IgG concentrations tested (p=7E-04, 8E-03 and 6E-03respectively). No inhibition was observed with the RA-AMA 1/E group IgGwas compared to the equivalent IgG concentration from the adjuvantcontrol. In order to determine if the inhibition caused by the IgG couldbe reversed, identical concentration of AMA1/E or RA-AMA 1/E proteinswere added to the culture during the GIA. The addition of 5.3 μg ml⁻¹AMA1/E protein to the 0.18 mg ml⁻¹ anti-AMA1/E IgG significantlyreverses inhibition compared to the addition of the same amount ofRA-AMA1/E (p=6E-03) (FIG. 6). This data indicates the critical role ofdisulphide bonds in the formation of epitopes that can induce inhibitoryanti-AMA-l antibodies.

[0150] ELISA, IFA and GIA data with antibodies to recombinant AMA1/E ,suggested that the conformational epitopes present on the refoldedprotein (in addition to the linear epitopes) were indeed highlyimmunogenic. A competition ELISA using sera from three rabbits in thelab-scale AMA1/E group (R-1, 2, 3) and two rabbits in the RA-AMA1/Egroup (R-7, 10) was done by pre-incubation with the 15 μg ml⁻¹ ofAMA1/E, RA-AMA1/E or with BSA as control. FIG. 6, shows the mean OD₄₀₅at 1:16,000 serum dilution for the two groups along with the SD for 3experiments. Pre-incubation with the refolded AMA1/E protein resulted onaverage 86% and 81% reduction in OD₄₀₅ in AMA1/E and RA-AMA1/E groupsrespectively, in comparison to the BSA control values in the same group(p=1.99E-13 and 1.55E-10 respectively). Although, RA-AMA1/E proteinpre-incubation resulted in an average 89% drop in the OD₄₀₅ in theRA-AMA1/E immunized group (p=5.63E-09), it resulted in an insignificant,16% drop in the refolded AMA1/E group (p=0.071). This data furthersuggests that a large proportion of the antibodies against refoldedAMA1/E were against disulphide bond dependent epitopes.

DISCUSSION

[0151] The availability of a significant quantity of a stablerecombinant protein having pharmaceutical levels of purity is anessential step on the path of testing adjuvant combinations capable ofinducing a long-lasting and high titer responses in humans. We describehere a process that was successfully scaled-up to produce AMA1/E , arecombinant protein based on the ectodomain of P. falciparum AMA-1. A300 L fermentation generated 4.5 kg of cell paste. Starting from 1.5 kgof cell paste 70 mg AMA1/E , enough material for over 800 doses, waspurified, vialed and lyophilized under a GMP environment. While thefinal yield of AMA1/E was relatively low, the AMA1/E product passed allthe major criteria set to proceed into a Phase I clinical trial. Theseinclude, purity (>99% done by SDS-PAGE and GPC), endotoxin content (0.06EU per 50 μg protein by LAL test), free thiol content (<0.1 μM free —SHgroups per μM protein measured by Ellman's test), positive reactivity toimmune reagents (monoclonal antibody 4G2dc1 and malaria immune sera doneby western blotting), mass analysis (54,648 Da by MALDI-TOF), correctN-terminal sequence (first 21 residues by Edman's method), host cellprotein content (<0.5% by ELISA), western blot (no E. coli specific bandwith up to 2 μg AMA1/E loaded per well), residual sarkosyl content(below detectable limits by RP-HPLC) and product stability (stable at37° C. for more than 6 months) in its lyophilized form (data not shown).The GMP produced AMA1/E product, was immunogenic in rabbits and raisedhigh titer inhibitory antibodies.

[0152] The correct folding of AMA1, as in case of several otherPlasmodium antigens, has been shown to be critical for its immunologicalactivity (Anders et al., 1998, supra; Hodder et al., 2001, supra;Crewther et al., 1996, supra). Full length P. falciparum AMA1 was firstexpressed in the eukaryotic insect cell system (Narum et al., 1993, J.Chromatogr. A. 657, 357-363), although the baculovirus product wassoluble, the purification strategy was not designed for scale-upproduction. Prokaryotic expression of AMA1 from various species has beenproblematic, primarily due to the formation of insoluble aggregatespresumably due to incorrect folding of the protein. Previous work on P.chabaudi AMA1 expression in E. coli showed that it was necessary toinclude an in vitro refolding step in the process in order to obtaincorrectly folded protein (Anders et al., 1998, supra). A similarapproach was successful for obtaining correctly folded AMA1 from P.falciparum and the antibodies made against it inhibited parasite growthin vitro (Hodder et al., 2001, supra). A scalable process for theproduction of recombinant AMA1 has not yet been described. Following thesuccess with another malarial antigen (Dutta et al., 2001, supra), weattempted to express r-AMA1/E as a soluble protein in E. coli. A ‘redoxmodified’ strain of E. coli, Origami (DE3), with mutations in theglutathione and thioredoxin reductase pathways (Bessette et al., 1999,Proc. Natl. Acad. Sci. USA 96, 13703-13708) was used for expression,with induction carried out at low temperature and using a minimal IPTGconcentration. Despite attempts to optimize the fermentation conditionsto obtain soluble r-AMA1/E , a large fraction was still located in theinsoluble pellet. Hence, a downstream purification process was developedto extract r-AMA1/E from both soluble and insoluble fractions and torefold it in vitro.

[0153] The use of Origami (DE3) cells was, in fact, a factor thatattributed to low protein yield due to plasmid loss during scale-upfermentation. Although the vector pWRMAL contained both ampicillin andtetracycline resistance genes for plasmid maintenance, penicillinderivatives like ampicillin cannot be used during the production of ahuman-use vaccine. The E. coli Origami (DE3) strain used to enhancesoluble protein expression carried a tetr gene, resulting in low plasmidmaintenance in the presence of tetracycline over the long growth periodrequired for scale-up fermentation. Future studies are being directed tothe selection of other bacterial hosts that allow the use of aselectable marker to increase plasmid maintenance.

[0154] The increase in reactivity to immune reagents observed after therefolding step and the homogeneity of the final product, justified theneed for the inclusion of this refolding step in the process, although,this was a limiting factor during scale up production. A minimum of40-fold dilution was necessary to gain optimal immune reactivity. Ananion-exchange step was used after refolding to separate the monomericAMA1/E from its aggregated forms, which eluted at a higher NaClconcentration. This monomer selection step resulted in some loss ofproduct during purification. After anion-exchange, a doublet at ˜10 kDa,was found to co-elute with AMA1/E and, although GPC was an option, weavoided it due to problems associated with scale-up. Instead, acation-exchange step using SP-Sepharose was used to purify AMA1/E tohomogeneity. Assays based on immuno-detection of HCP's, in combinationwith laser densitometry of stained polyacrylamide gels and analyticalGPC were used to determine that the final product was >99% pure.

[0155] N-terminal sequencing and mass spectrometric analysis confirmedthe correct primary structure of AMA1/E . Ellman's test and alkylationanalysis confirmed the absence of any free cysteines in the finalproduct. Shift in the RPC elution profile under reduced conditions andimmunoblot reactivity to a conformation dependent inhibitory ratmonoclonal antibody, 4G2dc1, under non-reduced conditions furtherconfirms the disulphide-bonded nature of the antigen.

[0156] AMA1/E was found to be highly immunogenic in rabbits incombination with Montanide ISA720 adjuvant. Antibodies raised againstthe recombinant protein recognized the native parasite AMA1 protein bothon IFA and western blot. Whole serum from the immunized rabbits showedgrowth inhibition of the homologous P. falciparum (3D7) parasites invitro both under suspension and static GIA conditions. During thisprocess development, we refolded the r-AMA1/E protein either directly(AMA1/E ) or after DTT reduction of the Ni⁺² column elution (RR-AMA1/E). The RR-AMA1/E product based on its overall lower immunogenicity,lower percent GIA values of its anti-sera, in addition to theobservation that DTT reduction gave no significant gain in the monomericprotein yield was not pursued further in the scale-up GMP process.

[0157] It has previously been shown that AMA1 based protective immunitycan be passively transferred by IgG transfusion into naive animals(Narum et al., 2000, supra; Anders et al., 1998, supra). Antibodies torecombinant AMA1 from P. falciparum have recently been reported toinhibit parasite invasion in vitro (Hodder et al., 1996, supra). Thesedata suggest that AMA1 based protection is probably antibody mediated.In vitro growth inhibition observed with whole sera and with purifiedIgG, in addition to the positive correlations observed between the ELISAtiter (against AMA1/E coated wells), and IFA titer, suggests that thesemeasures of antibody response might serve as good correlates of AMA1based protection in vivo. When comparing the same sera in parallelexperiments we have observed higher percent GIA values in static culturecompared to the suspension GIA (paired t test, p=5E-06). Fluid movementin suspension culture may better mimic the blood flow conditionsencountered in vivo by the parasite, than does static culture GIA. Someantibodies are known to show better inhibition in either static orsuspension culture (Haynes et al., 2002, supra) and it remains to beseen whether suspension or static culture GIA better predicts protectionfollowing vaccination. However, it is encouraging to report thatanti-AMA1/E antibodies are inhibitory under both conditions.

[0158] Previous data on mice vaccination with recombinant P. chabaudiAMA1 suggested that, the presence of intact disulphide bonds in thevaccinating AMA1 antigen are necessary to induce protection (Crewther etal., 1996, supra). The data presented here also suggests thatdisulphide-bond dependent motifs play a critical role in the inductionof inhibitory anti-AMA1 antibodies. Higher ELISA titer obtained withrefolded AMA1/E coated wells compared to the RA-AMA1/E coated wells inthe AMA1/E immunized group, lower IFA titers in the RA-AMA1/E group,inability of the anti-RA-AMA1/E antibodies to block parasite invasion,the ability of AMA1/E and not RA-AMA1/E protein to significantly reversethe in vitro growth inhibition and the ability of AMA1/E and not ofRA-AMA1/E, to out-compete binding of most of the anti-AMA1/E antibody toAMA1/E protein on ELISA, indicates that a majority of theimmunologically significant epitopes of AMA-1 are sensitive toreduction. In conclusion this application details the processdevelopment for the production of a disulfide cross-linked AMA1ectodomain recombinant protein that could serve as a malaria vaccinecandidate. Safety, stability and potency tests in animals are underway.

What is claimed is:
 1. A nucleotide fragment encoding P. falciparumAMA-1 ectodomain protein consisting of amino acids 83-531 of AMA-1. 2.The nucleotide fragment according to claim 1 wherein said fragment isfrom P. falciparum 3D7, Genbank Accession no. U65407.1.
 3. Thenucleotide fragment of claim 2, further comprising of a 6-histidine tagon the carboxy terminal end of the encoded protein.
 4. The nucleotidefragment of claim 3, further comprising of a 6-histidine tag on theamino terminal end of the encoded protein.
 5. The nucleotide fragment ofclaim 4, said fragment defined in SEQ ID NO:1.
 6. A recombinant vectorcomprising the nucleotide sequence of claim
 5. 7. The vector of claim 6wherein said vector is pWRMAL-AMA1/E .
 8. A recombinant vectorcomprising the nucleotide fragment of claim
 1. 9. An isolated P.falciparum AMA-1 ectodomain protein consisting of amino acids 83-531 ofAMA-1.
 10. An isolated AMA-1 protein according to claim 9 having theamino acid sequence defined in SEQ ID NO:2.
 11. A host cell transformedwith the vector according to claim
 7. 12. The host cell of claim 11wherein said host cytoplasm is oxidative.
 13. The host cell of claim 12wherein said host cell is Origami DE3.
 14. A method for isolating andpurifying recombinant P. falciparum AMA-1 protein comprising: growing ahost cell containing a recombinant vector expressing P. falciparum AMA-1protein according to claim 8 in a suitable culture medium, causingexpression of said vector under suitable conditions for production ofsoluble AMA-1 protein, lysing said host cells and recovering said AMA-1protein, and refolding said AMA-1 protein such that it reacquires itsnative folding.
 15. The method of claim 14 wherein said expression ofsaid vector is by induction with IPTG at a temperature range of 25°C.-30° C.
 16. The method of claim 14 wherein said induction is at 28° C.17. The method of claim 14 wherein lysing of cells is in the presence ofa mild detergent.
 18. The method of claim 17 wherein said mild detergentis sarkosyl.
 19. The method of claim 14 further comprising removal of E.coli proteins.
 20. The method of claim 19 wherein said removal of E.coli proteins is by application to a Ni-NTA column, followed by anionexchange chromatography, followed by cation exchange chromatography. 21.The method of claim 14 wherein said refolding is in the presence ofabout 1 mM reduced glutathione and about 0.25 mM oxidized glutathion.22. An isolated protein according to claim 9, wherein said proteinretains its native disulfide bridges.
 23. The isolated protein of claim22 wherein said purified protein is at least 95% pure.
 24. An isolatedprotein according to claim 22, wherein said purified protein is at least96% pure.
 25. An isolated protein according to claim 22 wherein saidpurified protein is at least 97% pure.
 26. An isolated protein accordingto claim 22 wherein said purified protein is at least 98% pure.
 27. Anisolated protein according to claim 22 wherein said purified protein isat least 99% pure.
 28. A method for in vitro diagnosis of malariaantibodies in a biological sample, comprising (i) contacting saidbiological sample with a composition comprising a AMA-1 proteinaccording to claim 22 under appropriate conditions which allow theformation of an immune complex, wherein said peptide is labeled with adetectable label, and (ii) detecting the presence of said immunecomplexes visually or mechanically.
 29. A kit for determining thepresence of malaria antibodies in a biological sample, comprising: atleast one peptide or protein composition according to claim 22, a bufferor components necessary for producing a buffer; means for detectingimmune complexes formed betweem the peptide and antibodies present inthe sample.
 30. A method for in vitro monitoring malaria infection orprognosing the response to treatment of patients suffering from malariainfection comprising: incubating a biological sample from a patient withmalaria infection with an AMA-1 protein according to claim 22 or asuitable part thereof under conditions allowing the formation of animmunological complex, removing unbound components, calculating theanti-AMA-1 titers present in said sample and monitoring the naturalcourse of malaria infection, or prognosing the response to treatment ofsaid patient on the basis of the amount anti-AMA-1 titers found in saidsample at the start of treatment and/or during the course of treatment.31. A kit for monitoring malaria infection or prognosing the response totreatment of patients suffering from malaria infection comprising: atleast one AMA-1 protein or a suitable part thereof according to claim22, a buffer or buffer components means for detecting the immunecomplexes formed between the peptide and antibodies present in thesample, and optionally, a means for determining the amount of immunecomplex formed.
 32. An antibody produced against the recombinant AMA-1protein according to claim
 22. 33. The antibody of claim 32 wherein saidantibody is monoclonal or polyclonal.
 34. A method for in vitrodiagnosis or detection of malaria antigen present in a biologicalsample, comprising: (i) contacting said biological sample with anantibody specific for the protein of claim 22, preferably in animmobilized form under appropriate conditions which allow the formationof an immune complex, (ii) removing unbound components, (iii) incubatingthe immune complexes formed with heterologous antibodies whichspecifically bind to the antibodies present in the sample to beanalyzed, with said heterologous antibodies conjugated to a detectablelabel under appropriate conditions, (iv) detecting the presence of saidimmune complexes visually or mechanically.
 35. A kit for in vitrodetection of a malaria antigen present in a biological sample,comprising: at least one antibody which reacts with the recombinantprotein of claim 22, wherein said antibody is preferentially immobilizedon a solid substrate, a buffer, or components necessary for producingthe buffer, enabling a binding reaction between these antibodies and themalaria antigens present in the biological sample, and a means fordetecting the immune complexes formed in the preceding binding reaction.36. An immunogenic composition comprising the isolated P. falciparumAMA-1 of claim
 22. 37. The composition of claim 36 further comprising anadjuvant.
 38. A vaccine against malaria comprising P. falciparum AMA-1according to claim
 22. 39. The vaccine of claim 38 further comprising anadjuvant.
 40. The vaccine of claim 39 wherein said adjuvant ismontanide.
 41. A method for inducing in a subject an immune responseagainst malaria infection comprising administering to said subject acomposition comprising an immunologically effective amount of P.falciparum AMA-1 of claim 22 in an acceptable diluent.
 42. The method ofclaim 41 wherein said composition further comprises an adjuvant.
 43. Themethod of claim 42 wherein said adjuvant is montanide.
 44. A method forinducing a protective immune response to malaria in a mammal, comprisingadministering a composition comprising a protein according to claim 22in an amount effective to induce an immune response in said mammal. 45.The method according to claim 44 wherein the composition furthercomprises an adjuvant.
 46. The method according to claim 45 wherein saidadjuvant is montanide.
 47. A multivalent vaccine for protection againstinfection with more than one strain of P. falciparum comprising a P.falciparum protein according to claim 22 from more than one strain of P.falciparum, said P. falciparum selected from the group consisting of3D7, FVO and CAMP.
 48. The multivalent vaccine of claim 47, furthercomprising an adjuvant.